Papilloma viruses are small DNA viruses that induce the hyper proliferation of epithelial cells. Approximately 70 different genotypes of human papilloma virus (HPV) have been isolated. Some HPV genotypes (e.g., 1, 2, 4, and 7) have been associated with human benign squamous papillomas (warts and condylomas) and others (e.g., 16 and 18) have been associated with human neoplastic and preneoplastic lesions (DiPaolo, et al., 1993, Crit. Rev. Oncogen. 4:337-360).
HPV-16 has been associated with a variety of clinical conditions in both women and men. In women, HPV-16 is frequently associated with latent infections, benign and premalignant cervical lesions (dysplasias/CIN) and half of invasive cervical carcinomas. Cervical cancer, which kills at least 500,000 women worldwide each year, proceeds through progressive cellular changes from benign condylomata to high-grade dysplasias/CIN before developing into an invasive cancer. In men, HPV-16 is associated with subclinical macular or clinical papular lesions. Penile Bowenoid papulosis which resembles carcinoma in situ. Detection and treatment of these lesions costs over five billion health care dollars annually in the United States.
HPV-16 has been associated with over half of invasive cervical carcinomas worldwide and with many cell lines derived from cervical carcinomas. HPV-16 expression causes benign proliferation and efficiently immortalizes cultured human epithelial cells, including cervical keratinocytes (DiPaolo, et al., 1993, Crit. Rev. Oncogen. 4:337-360; Zur Hausen & de Villiers, 1994, Annu. Rev. Microbiol. 48:427-447; Schiffman, 1995, J Natl. Cancer Inst. 87:1345-1347). Two HPV-16 genes, E6 and E7, and their gene products are required to immortalize a human keratinocytes and are a hallmark of cervical carcinoma (Hawley-Nelson et al., 1989, EMBO J. 8:3905-3910; Phelps et al., 1988, Cell 53:539-547; Viallet et al., 1994, Exp. Cell Res. 212:36-41; Yokoyama et al., Obstet. Gynecol. 83:197-204). The E6 and E7 proteins bind to other gene products (p53 and Rb tumor suppressors) to disrupt control of cell division and proliferation, leading to transformation (Scheffner et al., 1990, Cell 63:1129-1136; Zerfass et al., J. Virol 69:6389-6399).
Surgery is commonly used for treatment of high-grade lesions due to the lack of effective alternatives. Cervical laser ablation therapy, however, does not in the long term influence the natural history of cervical human papillomavirus-associated diseases in women. Interferons have not proved an effective antiviral or anticancer treatment. Chemotherapy (e.g., cisplatin, alone or combined with other chemotherapy agents such as 5FU) has generally not proved to be effective in treatment of many cervical cancers. Moreover, most chemotherapeutic agents are cytotoxic, leading to toxic side effects and the development of multiple drug resistance. Therefore, there is a need for reagents than can specifically inhibit the growth of HPV-associated tumor cells, while avoiding serious toxic reactions.
HPV-specific treatments in the form of cleavage of HPV-specific RNA with ribozymes and inhibition by HPV-specific antisense oligonucleotides have been suggested (PCT International Patent Application WO 95/31552; DiPaolo, et al., 1993, Crit. Rev. Oncogen.4:337-360; Steele, et al., 1993, Cancer Res. 53:2330; Storey, et al., 1991, Nuc. Acids Res. 19(15):4109). Ribozymes are small catalytic RNA molecules that can hybridize to and cleave a complementary RNA target (Cech, 1988, JAMA 260:3030-3034). Ribozymes having a "hairpin" motif have been found to be more efficient than the "hammerhead" motif (Hampel & Tritz, 1989, Biochem. 28:4929-4933; Hampel, et al., 1990, Nuc. Acids Res. 18:299-304) and "hairpin" ribozymes have been used to cleave viral targets, including the human immunodeficiency virus (HIV-1) and HPV (Ojwang, et al., 1992, Proc. Natl. Acad. Sci. USA 89, 10802-10806; Yu, et al., 1993, Proc. Natl. Acad. Sci. USA 90:6340-6344; PCT International Patent Application WO 95/31552).
Antisense RNA and oligonucleotides hybridize to complementary mRNA, thus blocking translation and promoting the activity of endogenous RNase H to cleave the mRNA (Walder, 1988, Genes Dev. 2:502-504; Cohen, 1991, Antisense Res. Dev. 1:191-193). Although antisense RNA and oligonucleotides should be specific for their target sequence, nonspecific toxicity has been observed (Henry et al., 1997, Toxicol. 116:77-88; Henry et al., 1997, Anticancer Drug Des. 12:1-14). First-generation antisense phosphorothioates, whose nucleotide backbones carry sulfur atoms to slow intracellular degradation were often ineffective because of inability to enter cells or to complement the target mRNA, but improved second generation phosphorothioate antisense therapies, referred to as "mixed backbone oligonucleotides" and "end-modified chimerics" that carry 2'-O-methylribonucleoside moieties have proven effective in clinical trials (Monia et al., 1996, Nature Medicine 6:668-675; Roush, 1997, Science 276:1192-1193; Agrawal et al., 1997, Proc. Natl. Acad. Sci. USA 94:2620-2625; Agrawal, 1996, TIBTECH 14:3-14).
Antisense inhibition of HPV-18 E6 and E7 expression in cell lines (C4-1 and HeLa) resulted in a significant decrease in growth rate with continuous addition of oligonucleotide (Steele, et al., 1993, Cancer Res. 53:2330-2337). Similar results have been observed in cells transfected with recombinant vectors (von Knebel Doeberitz & Grissmann, 1987, Hamatol. Bluttransfus. 31:377-279; Hamada et al., 1996, Gynecol. Oncol. 63:219-227).
The present invention discloses oligonucleotide sequences and methods of antisense therapy using antisense oligonucleotides defined by selected HPV-16 complementary sequences.